ARTICLE

DOI: 10.1038/s41467-017-02532-x OPEN Auditory opportunity and visual constraint enabled the evolution of echolocation in bats

Jeneni Thiagavel1, Clément Cechetto 2, Sharlene E. Santana3, Lasse Jakobsen2 Eric J. Warrant 4 & John M. Ratcliffe 1,2,5,6

Substantial evidence now supports the hypothesis that the common ancestor of bats was nocturnal and capable of both powered flight and laryngeal echolocation. This scenario entails

1234567890():,; a parallel sensory and biomechanical transition from a nonvolant, vision-reliant mammal to one capable of sonar and flight. Here we consider anatomical constraints and opportunities that led to a sonar rather than vision-based solution. We show that bats’ common ancestor had eyes too small to allow for successful aerial hawking of flying insects at night, but an auditory brain design sufficient to afford echolocation. Further, we find that among extant predatory bats (all of which use laryngeal echolocation), those with putatively less sophis- ticated biosonar have relatively larger eyes than do more sophisticated echolocators. We contend that signs of ancient trade-offs between vision and echolocation persist today, and that non-echolocating, phytophagous pteropodid bats may retain some of the necessary foundations for biosonar.

1 Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, ON M5S 3B2, Canada. 2 Department of Biology, University of Southern Denmark, Campusvej 55, 5230, Odense C, Denmark. 3 Department of Biology and Burke Museum of Natural History and Culture, University of Washington, Seattle, WA 98195, USA. 4 Department of Biology, Lund University, Sölvegatan 35, 22362 Lund, Sweden. 5 Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada. 6 Department of Natural History, Royal Ontario Museum, 100 Queens Park, Toronto, ON M5S 2C6, Canada. Correspondence and requests for materials should be addressed to J.M.R. (email: [email protected])

NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x

ats (Chiroptera) are the second largest order of mammals, relative to ancestral states (ASs). These three hypotheses reflect comprising >1300 species and characterized by powered mechanistic explanations for the origination and evolution of LE Bfl 1 2 fl ight . The vast majority of bats are strictly nocturnal , in bats for pursuing ying insects, and predict auditory oppor- with a few species also active around dawn and dusk3,4. Long tunity and visual constraint5,35. Specifically, we test predictions before the discovery of echolocation, bats were once divided into that the ancestral bat had (i) an auditory brain design capable of “megabats” (members of the family Pteropodidae) and “micro- supporting early LE, but (ii) eyes of insufficient absolute size to – bats” (the remaining ~20 chiropteran families)1,5 7. Today these allow insect tracking at night. We also test the predictions that are anachronistic terms, and the pteropodids [(~200 vision- today not only would pteropodids possess relatively larger eyes dependent species, none using laryngeal echolocation (LE)] are than LE bats but that among predatory bats (all of which use LE), placed in Yinpterochiroptera (a.k.a. Pteropodiformes), which (i) MH bats would have relatively larger eyes than DH and CF together with Yangochiroptera (a.k.a. Vespertilioniformes) com- bats and (ii) short-wavelength-sensitive (SWS) opsin genes would prise the two chiropteran suborders. Both suborders are otherwise remain functional in MH and DH bats, but have lost functionality comprised solely of laryngeal echolocators6,7. From a strict par- in CF species36. simony perspective, LE, if considered as a single trait, could Using modern phylogenetic comparative methods and a recent therefore have evolved once in bats, and subsequently been lost in bat molecular phylogeny, we find support for each of these four the pteropodids. Alternatively, LE could have evolved at least hypotheses. Specifically, our analyses unambiguously support the twice independently, once or more in Yangochiroptera, and once idea that the common ancestor of modern bats was a small, flying or more in Yinpterochiroptera, after the pteropodids diverged6,8 nocturnal predator capable of LE and that this complex sensory (Fig. 1). The sum of evidence, however, indicates (i) that the bats’ trait has regressed in the pteropodids. Further, our results suggest common ancestor was a predatory laryngeal echolocator and (ii) that this vocal–auditory solution was favored over vision due to that the phytophagous pteropodids have lost most, but perhaps – not all, hallmarks of this complex active sensory system8 12. 5,13 Since Donald Griffin’s discovery of echolocation , the pre- Pteropodidae vailing view has been that the first bats were small, nocturnal, – insectivorous echolocators6,14 16. Specifically, bats are thought to – have originated ≥64 mya17,18 to exploit the then unrealized foraging niche of small, night-flying insects, a resource most bats rely on today5,12,15,19. In darkness and dim light, nascent echo- location would have allowed these bats to pursue these then Rhinolophoidea vulnerable insects5,15. Griffin5 too divided bats into those that use laryngeal echolocation (hereafter, LE bats) and those that do not + (i.e., the pteropodids). Within LE bats, he identified three groups: (i) bats producing multi-harmonic (MH) calls, with little fre- + quency modulation, (ii) bats producing downward-sweeping, Emballonuroidea frequency-modulated calls, with most energy in the fundamental harmonic, and (iii) bats producing long, constant frequency calls, with energy typically concentrated in the second harmonic5. These four sensory divisions—non-laryngeal echolocators (hereafter, pteropodids or NLE bats), multi-harmonic echoloca- tors (MH bats), frequency modulating, dominant harmonic + Noctilionoidea echolocators (DH bats), and constant frequency echolocators (CF bats)—remain robust functional descriptors of biosonar diversity – in bats6,20. Griffin5 and others since6,12,14,21 24 have suggested that MH calls most closely reflect bats’ ancestral condition. If so, pteropodids, DH, and CF bats would represent three derived sensory states6,10,12. While no pteropdodid is thought to be Vespertilionoidea predatory or to be capable of LE, members of the genus Rousettus use biosonar based on tongue clicking for orientation25,26. This form of echolocation, while effective, falls short of the maximum detection distances and update rates observed in LE bats19,25,26. Among LE bats, it has been previously argued that CF and DH bats possess more advanced abilities for detecting and tracking Fig. 1 Two equally parsimonious hypotheses for the origination of laryngeal – flying prey than do MH bats5,27 30. Similarly, different visual echolocation in bats. The unshaded side depicts the two origins hypothesis abilities exist within today’s bats, with pteropodids possessing the and predicts that laryngeal echolocation originated in the common ancestor most advanced visual systems, in some species including an optic to the Emballonuroidea, Noctilionoidea, and Vespertilionoidea and again in chiasm31, and the LE vespertilionids and rhinolophids perhaps the Rhinolophoidea. The shaded side depicts the single origin hypothesis, the least32. Interestingly, all rhinolophids use CF calls, while most which predicts laryngeal echolocation was present in the common ancestor vespertilionids use DH calls12,20,29. It is among the MH species of all bats and lost in the Pteropodidae. Middle column displays (top to that we find the LE bats capable of the greatest quantified visual bottom) five 30–35 g species from each of these major groups: Cynopterus resolution32,33 and even ultraviolet light sensitivity34. brachyotis (non-echolocating, phytophagous), Rhinolophus hildebrandti Here, we use phylogenetic comparative methods to further test (echolocating, predatory), Taphozous melanopogon (echolocating, the hypothesis that the ancestral bat was a small, predatory predatory), Tonatia evotis (echolocating, predatory), Nyctalus noctula echolocator, which produced MH biosonar signals5,12,14. Addi- (echolocating, predatory). Please note that bats with constant frequency tionally, we test three hypotheses about the relationships between (CF), multi-harmonic frequency-modulated calls (MH) and fundamental visual abilities and echolocation behavior in bats across their four harmonic frequency modulated calls (DH) (i.e., most energy in fundamental sensory divisions, and with respect to diet and roosting behavior, harmonic) are found in both suborders of bats. Photographs by Brock Fenton and Signe Brinkløv 2 NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x ARTICLE pre-existing sensory opportunities and constraints and that these R2 = 0.879). Thus, we generated phylogenetic residuals for log- sensory trade-offs persist to different extents in bats today. transformed brain mass, eye size, and brain region masses on body mass, and tested for differences in these residuals across our Results three foraging strategies and four biosonar signal designs. Estimated states and traits of the ancestral bat. We estimated Bayesian posterior probabilities for both foraging strategy and call Ancestral brain regions versus modern foraging categories. type under an equal rates (EQ) model of evolution, as this model Phylogenetic analyses of variance (ANOVAs) indicate that pter- produced the lowest AICc scores for both categories (foraging fi = = = opodid bats are signi cantly larger than animal-eating bats (F category: EQ AICc 45.43, symmetric transition (SYM) AICc 40.353, p = 0.03; Supplementary Table 1). We also found that 48.51, all-rates-different (ARD) AICc = 53.35; call design cate- = = = absolute brain, neocortex, hippocampus, and olfactory bulb sizes gories: EQ (AICc) 41.2, SYM 44.17, ARD 49.44). We found are significantly larger in pteropodids than in animal-eating bats an additional support for the hypothesis that the ancestral bat was (brain: F = 70.763, p = 0.009; neocortex: F = 55.618, p = 0.006; a predatory, echolocating bat (Bayesian posterior probabilities: hippocampus: F = 82.641, p = 0.001; olfactory bulb: F = 85.068, p animal-eating laryngeal echolocator >0.999, phytophagous lar- = < < 0.001; Supplementary Table 1). AS reconstructions suggest that yngeal echolocator 0.001, phytophagous NLE 0.001; Fig. 2a). these structures have become larger in pteropodids, while the These same results support the idea that phytophagy has origi- neocortex and olfactory bulb may have become smaller in nated at least twice in the Chiroptera, once in the Pteropodidae animal-eating species (Fig. 3; Supplementary Fig. 1). We found and at least once in the Phyllostomidae (Fig. 2a). With respect to that absolute superior colliculi are larger in pteropodid bats than biosonar signal design, we found support for the hypothesis that in animal-eating bats (F = 26.937, p = 0.014; Supplementary the ancestral bat produced multiharmonic calls (Bayesian pos- > < Table 1) and larger in pteropodids compared to ancestral terior probabilities: MH 0.999, constant frequency 0.001, reconstructions, suggesting greater investment in visual tracking fundamental harmonic frequency modulated <0.001; non- < (Fig. 3; Supplementary Fig. 1). For each of these traits, the phy- laryngeal echolocating: 0.001; Fig. 2b). tophagous phyllostomids fell somewhere between the pteropodids We reconstructed ASs of body and brain mass. These and animal-eating bats, and did not differ from either of these reconstructions suggest that the ancestral bat was ~20 g, roughly groups significantly (Supplementary Table 1). ’ – the mean size of today s laryngeal echolocating bats, and smaller We, like previous authors40 42, found that phytophagous than most extant pteropodid bats (Fig. 3; Supplementary Fig. 1), > species in general have relatively (i.e., phylogenetically informed with a relative brain mass 20% smaller than that of extant mass-residuals) larger brains (F = 113.747, p = 0.001) than pteropodid species (body mass: N = 183, root AS = 18.55 g, 95% fi = predatory bats. Also, like previous studies, we found that con dence interval (CI) 7.18 (lower limit), 47.91 (upper limit); phytophagous species have relatively larger neocortices (F = brain mass: N = 183; AS = 428.33 mg, CI = 229.59, 799.08), = = = fi 37 80.525, p 0.002), hippocampi F 126.534, p 0.001), olfactory con rming a previous report . For comparison with AS bulbs F = 129.473, p = 0.001) than do predatory species41,43. reconstructions of auditory brain regions (see below) and for Additionally, we found that the phytophagous bats also had comparison with modern day bats, we also reconstructed the ASs relatively larger superior colliculi (F = 35.649, p = 0.006) than of several non-auditory brain region masses associated with animal-eaters (Supplementary Table 2). sensory information processing (neocortex: N = 149; AS = 94.15 mg, CI = 61.25, 144.71; hippocampus: N = 149; AS = 26.53 mg, CI = 18.24, 38.58; olfactory bulb: N = 149; AS = 9.02 mg, CI = 5.8, Ancestral brain regions versus modern echolocation categories. 14.05; superior colliculus: N = 84; AS = 6.66 mg, CI = 4.69, 9.45; With respect to echolocation signal design, we found that bats Fig. 3; Supplementary Fig. 1). that do not produce echolocation signals using their larynges (i.e., the pteropodids) had larger absolute brains, neocortices, hippo- campi and olfactory bulbs than did CF and DH bats (brain: F = Phylogenetic signal. To estimate the degree to which phylogeny = = = predicts the pattern of covariance among species, we used a recent 49.96, p 0.016, neocortex: F 41.58, p 0.016, hippocampus: F 38 ’ 39 = 43.16, p = 0.017, olfactory bulb: F = 35.09, p = 0.025) and larger molecular phylogenetic tree and Pagel s lambda . We found = = significant phylogenetic signal for all log-transformed variables absolute superior colliculi than DH bats (brain: F 21.36, p (body mass: λ = 0.9787; logL = −210.37, p < 0.001; brain mass: λ = 0.011) (see Supplementary Table 3). 0.82; logL = −143.59, p < 0.001; eye mass: λ = 0.95; logL = −351.73, In relative terms, we found that the pteropodids had larger < = − < λ = = relative brains, neocortices, olfactory bulbs than both CF and DH p 0.001; logL 55.85, p 0.001; neocortex: 0.95; logL = = = = −135.26, p < 0.001; hippocampus: λ = 0.92; logL = −187.85, p < bats (brain: F 84.54, p 0.002, neocortex: F 90.16, p 0.001, λ = = − < olfactory bulb: F = 25.45, p = 0.016), larger relative hippocampi 0.001; superior colliculus: 1.0; logL 72.65, p 0.001; olfac- = = tory bulb: λ = 0.97; logL = −146.76, p < 0.001; inferior colliculus: λ than DH bats (F 35.82, p 0.023), and larger relative superior = = − < λ = = colliculi than all laryngeal echolocating bats, regardless of call 0.87; logL 72.79, p 0.002; auditory nucleus: 0.84; logL = = −68.21, p < 0.001). type (F 44, p 0.001). Other than with respect to relative superior colliculus size, MH bats fell between the pteropodids, on the one hand, and the CF and DH bats, on the other, for all other Mass-residuals. We show (using phylogenetic generalized least- measures of relative brain and brain region size (see Supplemen- squares by restricted maximum likelihood) that brain and eye tary Table 4). mass were positively correlated with body mass (brain: b = 0.654 ± 0.010; t = 64.917; p < 0.001; R2 = 0.877; eye: b = 0.745 ± 0.056; t = 13.197; p < 0.001; R2 = 0.705), as were brain regions (inferior Ancient auditory brain versus modern foraging categories. colliculus: b = 0.566 ± 0.030; t = 18.673 p < 0.001, R2 = 0.295; With respect to relative auditory brain region size, we found the auditory nucleus: b = 0.578 ± 0.000; t = 745,121.1, p < 0.001, R2 = opposite trends to those above for the inferior colliculi and 0.246; superior colliculus: b = 0.590 ± 0.021; t = 28.344, p < 0.001, auditory nuclei. Specifically, pteropodid auditory brain regions R2 = 0.930; olfactory bulb: b = 0.726 ± 0.035; t = 20.895, p < 0.001, were relatively smaller than those of laryngeal echolocating bats41 R2 = 0.800; hippocampus: b = 0.603 ± 0.022; t = 26.896, p < 0.001, (inferior colliculus: F = 73.291, p = 0.001; auditory nucleus: F = R2 = 0.740; neocortex: b = 0.710 ± 0.000; t = 9,202,949, p < 0.001, 58.585, p = 0.001; Supplementary Table 2).

NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications 3 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x

Otomops martiensseni Chaerephon jobensis

Chaerephon pumilus Cheiromeles torquatus a Tadarida aegyptiaca

Natalus tumidirostris condylurus Mops

Sphaeronycteris toxophyllum Artibeus jamaicensis

Artibeus lituratus Artibeus concolor Molossus ater

Artibeus glaucus

Artibeus toltecus Artibeus phaeotis

Platyrrhinus brachycephalus Platyrrhinus infuscusArdops nichollsi

Molossus molossus Eumops auripendulus Eumops perotis Platyrrhinus vittatus Eumops glaucinus Miniopterus schreibersii Artibeus hartii Miniopterus magnater Miniopterus tristis Miniopterus pusillus Miniopterus inflatus Vampyrodes caraccioli Myotis mystacinus Platyrrhinus helleri Myotis lucifugus Myotis riparius Myotis nigricans Platyrrhinus lineatus Myotis albescens Chiroderma villosum Myotis tricolor Myotis bocagii Chiroderma trinitatum Myotis daubentonii Myotis bechsteinii Mesophylla macconnelli Myotis myotis Chiroderma salvini Myotis nattereri Myotis muricola Uroderma bilobatum Myotis dasycneme Myotis horsfieldii Myotis adversus Sturnira tildae Myotis annectans Sturnira ludovici Scotophilus kuhlii Scotophilus heathii RhinophyllaSturnira pumilio lilium Scotophilus nigrita Carollia perspicillata Scotophilus dinganii Lasiurus borealis Carollia castanea Rhogeessa parvula Lionycteris spurrelli Rhogeessa tumida Lonchophylla mordax Pipistrellus subflavus Lonchophylla thomasi Barbastella barbastellus Plecotus auritus Phyllostomus hastatus Phyllostomus elongatus Eptesicus fuscus Eptesicus brasiliensis Mimon crenulatum Eptesicus serotinus Phylloderma stenops Pipistrellus javanicus Nyctalus noctula Lophostoma schulzi Pipistrellus kuhlii Lophostoma silvicolum Vespertilio murinus Tonatia bidens Tylonycteris pachypus Phyllostomus diacolor Tylonycteris robustula Chalinolobus gouldii Trachops cirrhosus Chalinolobus morio Macrophyllum macrophyllum Cynopterus sphinx Vampyrum spectrum Cynopterus brachyotis Glossophaga longirostris Megaerops niphanae Glossophaga soricina Megaerops ecaudatus Scotonycteris zenkeri Monophyllus plethodon Rousettus leschenaultii Brachyphylla cavernarum Rousettus aegyptiacus Anoura geoffroyi Myonycteris torquata Anoura caudifer Hypsignathus monstrosus Epomops franqueti Micronycteris megalotis Epomophorus wahlbergi Micronycteris minuta Micropteropus pusillus Epomophorus gambianus Micronycteris brachyotis Syconycteris australis Pteronotus personatus Pteronotus devyi Macroglossus minimus Paranyctimene raptor Pteropus samoensis Pteronotus gymnonotus Pteronotus parnellii Pteropus tonganus Pteropus hypomelanus Pteropus poliocephalus MormoopsThyroptera megalophylla tricolor Pteropus vampyrus Noctilio leporinus Megaderma spasma Noctilio albiventris Cardioderma cor Rhinopoma microphyllum Furipterus horrens Triaenops persicus Hipposideros commersoni Peropteryx macrotis Hipposideros armiger Peropteryx trinitatis Hipposideros turpis Hipposideros diadema Cormura brevirostris Hipposideros ruber Hipposideros caffer Hipposideros galeritus Hipposideros cervinus SaccopteryxSaccopteryx canescens bilineata Hipposideros fulvus Hipposideros ater Saccopteryx leptura Hipposideros speoris Rhinolophus affinis Coleura afra Rhinolophus stheno Rhinolophus lepidus Rhinolophus megaphyllus Rhinolophus malayanus Rhinolophus trifoliatus

Rhynchonycteris naso Rhinolophus hipposideros Rhinolophus luctus Rhinolophus landeri

Emballonura monticola

Emballonura semicaudata Taphozous mauritianus Nycteris arge

Nycteris javanica Taphozous melanopogon Nycteris grandis TaphozousTaphozous hildegardeae longimanus Nycteris macrotis Nycteris hispida Nycteris thebaica

Saccolaimus saccolaimus Predatory laryngeal echolocators (>0.999)

Rhinolophus clivosus

Rhinolophus eloquens Rhinolophus capensis Phytophagous laryngeal echolocators (<0.001) Rhinolophus hildebrandti

Rhinolophus ferrumequinum Phytophagous non-laryngeal echolocators (<0.001)

Otomops martiensseni

Chaerephon jobensis b Chaerephon pumilus Cheiromeles torquatus Tadarida aegyptiaca

Natalus tumidirostris condylurus Mops

Sphaeronycteris toxophyllum Artibeus jamaicensis

Artibeus lituratus Artibeus concolor Molossus ater

Artibeus glaucus Artibeus toltecus Artibeus phaeotis

Platyrrhinus brachycephalus Platyrrhinus infuscusArdops nichollsi

Molossus molossus

Eumops auripendulus Eumops perotis Eumops glaucinus Platyrrhinus vittatus Miniopterus schreibersii Artibeus hartii Miniopterus magnaterMiniopterus tristis Miniopterus pusillus Miniopterus inflatus Myotis mystacinus Vampyrodes caraccioli Myotis lucifugus Platyrrhinus helleri Myotis riparius Myotis nigricans Platyrrhinus lineatus Myotis albescens Myotis tricolor Chiroderma villosum Myotis bocagii Chiroderma trinitatum Myotis daubentonii Myotis bechsteinii Myotis myotis MesophyllaChiroderma macconnelli salvini Myotis nattereri Myotis muricola Uroderma bilobatum Myotis dasycneme Myotis horsfieldii Myotis adversus Sturnira tildae Myotis annectans Sturnira ludovici Scotophilus kuhlii Scotophilus heathii RhinophyllaSturnira pumilio lilium Scotophilus nigrita Carollia perspicillata Scotophilus dinganii Lasiurus borealis Carollia castanea Rhogeessa parvula Lionycteris spurrelli Rhogeessa tumida Lonchophylla mordax Pipistrellus subflavus Lonchophylla thomasi Barbastella barbastellus Phyllostomus hastatus Plecotus auritus Phyllostomus elongatus Eptesicus fuscus Eptesicus brasiliensis Mimon crenulatum Eptesicus serotinus Phylloderma stenops Pipistrellus javanicus Nyctalus noctula Lophostoma schulzi Pipistrellus kuhlii Lophostoma silvicolum Vespertilio murinus Tonatia bidens Tylonycteris pachypus Phyllostomus diacolor Tylonycteris robustula Trachops cirrhosus Chalinolobus gouldii Chalinolobus morio Macrophyllum macrophyllum Cynopterus sphinx Vampyrum spectrum Cynopterus brachyotis Glossophaga longirostris Megaerops niphanae Glossophaga soricina Megaerops ecaudatus Scotonycteris zenkeri Monophyllus plethodon Rousettus leschenaultii Brachyphylla cavernarum Rousettus aegyptiacus Anoura geoffroyi Myonycteris torquata Anoura caudifer Hypsignathus monstrosus Epomops franqueti Micronycteris megalotis Epomophorus wahlbergi Micronycteris minuta Micropteropus pusillus Epomophorus gambianus Micronycteris brachyotis Syconycteris australis PteronotusPteronotus personatus devyi Macroglossus minimus Paranyctimene raptor Pteropus samoensis Pteronotus gymnonotus Pteronotus parnellii Pteropus tonganus Pteropus hypomelanus Pteropus poliocephalus MormoopsThyroptera megalophylla tricolor Pteropus vampyrus Noctilio leporinus Megaderma spasma Noctilio albiventris Cardioderma cor Rhinopoma microphyllum Furipterus horrens Triaenops persicus Hipposideros commersoni Peropteryx macrotis Hipposideros armiger Peropteryx trinitatis Hipposideros turpis Hipposideros diadema Cormura brevirostris Hipposideros ruber Hipposideros caffer Hipposideros galeritus Hipposideros cervinus Saccopteryx canescens Hipposideros fulvus Saccopteryx bilineata Hipposideros ater Saccopteryx leptura Hipposideros speoris Rhinolophus affinis Coleura afra Rhinolophus stheno Rhinolophus lepidus Rhinolophus megaphyllus Rhinolophus malayanus Rhynchonycteris naso Rhinolophus trifoliatus Rhinolophus luctus

Rhinolophus hipposideros Rhinolophus landeri Emballonura monticola

Emballonura semicaudata Taphozous mauritianus Nycteris arge

Nycteris javanica

Taphozous melanopogon Nycteris grandis TaphozousTaphozous hildegardeae longimanus Nycteris macrotis Nycteris hispida Nycteris thebaica MH bats (–99.9%) Saccolaimus saccolaimus NLE bats (<0.0004%)

Rhinolophus clivosus

Rhinolophus eloquens

Rhinolophus capensis Rhinolophus hildebrandti DH bats (<0.004%)

Rhinolophus ferrumequinum CF bats (<0.005%)

4 NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x ARTICLE

However, we found no differences between foraging categories MH, DH, and CF predatory bats with respect to absolute body, with respect to the absolute masses of the auditory brain regions: brain, brain region, nor eye mass (i.e., all traits considered; absolute inferior colliculus size (F = 0.323, p = 0.946) and absolute Supplementary Table 5). However, we found differences among auditory nucleus size (F = 0.043, p = 0.992), which remain similar these bats with respect to relative eye mass (F = 31.450, p = 0.048), across these three categories (Supplementary Table 1). Neither neocortex mass (F = 41.499, p = 0.004), and superior colliculus did we observe any differences in the sizes of auditory brain mass (F = 14.256 p = 0.048) (Fig. 4; Supplementary Table 6). regions in modern bats relative to the common ancestor (inferior Specifically, we found that relative eye size was significantly colliculus: N = 84; AS = 12.93 mg, CI = 9.09, 18.4; auditory larger in MH bats than in DH bats (p = 0.05), and nearly so in nucleus: N = 84; AS = 5.7 mg, CI = 4.08, 7.97) (Fig. 3; Supplemen- MH versus CF bats (p = 0.08). We found no difference in relative tary Fig. 1; Supplementary Table 1). eye size between DH and CF bats (p = 0.957). Similarly, we found that relative neocortex was larger in MH than in DH bats (p = Ancient auditory brain versus modern echolocation categories. 0.003), and nearly so with respect to relative superior colliculus mass (p = 0.063) (Fig. 4; Supplementary Table 6). Last, as With respect to biosonar signal design categories, we found that 36 there were no significant differences in the absolute auditory predicted , we found that the ancestral bat likely possessed regions of the non-laryngeal echolocating pteropodids and any of functional SWS opsin genes (Bayesian posterior probabilities: the laryngeal echolocating bats, regardless of signal design functional SWS opsin gene: 0.971; non-functional SWS opsin (inferior colliculus: F = 1.558, p = 0.826, auditory nucleus: F = gene: 0.029), and that among predatory bats, these genes are now = non-functional in CF bats but remain functional in MH and DH 1.558, p 0.817; Supplementary Table 3). In relative terms, we = found that the pteropodids had smaller auditory regions than bats (p 0.002) (Supplementary Fig. 3). Because the above result surprised us, we wanted to consider laryngeal echolocating bats, regardless of call type (inferior col- fl liculus: F = 42.598, p = 0.001, auditory nucleus: F = 47.309, p = the potential in uence of roosting preference (i.e., whether light 0.001) (see Supplementary Table 4). environment might impact relative eye size). However, after categorizing predatory species as roosting either exclusively in caves/cavities or as also using exposed roosts (Supplementary Eye size in the ancestral bat versus in modern bats. We found Data 1), we found no differences in absolute (F = 0.676, p = 0.427) that the Plasticine models best predicted eye diameters reported or relative (F = 0.412, p = 0.545) eye mass between the 3 call type in the literature, and thus used these as proxies for eye diameter groups. We also found no significant relationship between these 2 = < in our analyses (Plasticine model: R 0.9, p 0.001; eyelid two roost categories and ancestral (MH) versus derived (CF + 2 = < – 2 = < length: R 0.78, p 0.001; ZB IOD: R 0.3, p 0.002). Using DH) call types (p = 0.950). We did however find that the ancestral = these estimates, we reconstructed the AS of absolute eye size (N bat was likely to have roosted using exposed surfaces, rather than = = − 183; AS 7.67 mg, CI 20.23, 110.09; Fig. 3). This translates caves or cavities (Bayesian posterior probabilities: exposed roosts: into an ancestral eye diameter of 3.13 mm. This is smaller than 0.998, caves/cavities: 0.002). the smallest pteropodid eye found today (Syconycteris australis Figure 5 illustrates the phylogenetically informed linear with a diameter of 5.03 mm) and similar in size to that of the regressions between eye and body mass in the five most speciose largest extant phytophagous and predatory laryngeal echolocating families of bats, and suggests the strictly predatory emballonurids bats (see Supplementary Data 1). (all MH bats) have the largest eyes among LE bats. We also confirmed that pteropodids have absolutely larger eyes than do laryngeal echolocators (F = 149.248, p = 0.001; Supple- mentary Table 1), and compared to the AS estimate at the root Discussion node, this suggests a trend of increasing eye size in pteropodids Our comparative analyses lend strong support to the already well- and possible reduction in eye size in most extant laryngeal supported hypothesis that the common ancestor of bats was a echolocating bats (Fig. 3; Supplementary Fig. 1). We also found small (~20 g), predatory, laryngeal echolocator5,12,37. Specifically, that in relative terms, the non-laryngeal echolocating pteropodids a bat that took flying insects on the wing at night5 and roosted had larger eyes than laryngeal echolocating bats, regardless of diet externally, rather than deep in caves. Our results, thus, also (F = 88.362, p < 0.001) (Supplementary Table 2) or call type support the conclusion that LE has been lost, rather than never (absolute: F = 136.18, p = 0.001; relative: F = 146.86, p = 0.001) (see gained, in the family Pteropodidae8 (Figs. 1–3). They also indicate Supplementary Tables 3 and 4). that a switch to a phytophagous diet occurred at least twice in To consider the relationship between visual investment and bats since their origin, once in the pteropodids (Yinpterochir- echolocation sophistication, without the confounding effects of optera) and once or more within the laryngeal echolocating diet, we then considered the relationships between all traits and family Phyllostomidae (Yangochiroptera)44 (Fig. 2a). echolocation call design in only predatory species (i.e., after We also confirm relative brain size is greater in phytophagous removing all pteropodids and all phytophagous phyllostomids). bats (i.e., the pteropodids and the laryngeal echolocating phyto- – That is, between predatory bats producing MH calls (AS), and CF phagous phyllostomids) than today’s predatory bats40 42,45 and DH calls (both derived). We found no differences between (Supplementary Table 2; Supplementary Fig. 2), and compared to

Fig. 2 The ancestral state estimates of call types and foraging categories. a The echolocation signals of bat species (N = 183) were categorized as (i) constant frequency (CF), (ii) multi-harmonic calls (MH), (iii) frequency modulated calls dominated by the fundamental harmonic (DH), or non-laryngeal (NLE, i.e., pteropodids). Models of evolution were compared using AICc scores and the character states for ancestral call types were estimated under an equal rates model of evolution. These marginal ancestral states (i.e., the empirical Bayesian posterior probabilities) have been overlain on the phylogeny. We find support for a multi-harmonic ancestral call type (Bayesian posterior probabilities: CF: <0.001; MH: >0.999; DH: <0.001; MLE: <0.001). b Bats were also categorized as (i) predatory laryngeal echolocators (ALE), (ii) phytophagous laryngeal echolocators (PLE) and (iii) phytophagous non-laryngeal echolocators (PNLE). Models of evolution were compared using AICc scores and the character states for ancestral call types were estimated under an equal rates model of evolution. These marginal ancestral states have been overlain on the phylogeny. Our results suggest that the ancestral bat was a predatory laryngeal echolocator (Bayesian posterior probabilities: ALE: >0.999; PLE: <0.001; PNLE: <0.001)

NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications 5 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x ab c 250 1600 500 1400 200 1200 400 150 1000 300 800 Body (g)

100 Eye (mg) 200 600 Neocortex (mg) 400 50 100 200

0 0 0 ALE PLE PNLE ASR ALE PLE PNLE ASR ALE PLE PNLE ASR de f 15 60 30 12.5 50 25 10 40 20

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20 10 5 Auditory nucleus (mg) Inferior colliculus (mg) Superior colliculus (mg) 10 5 2.5

0 0 0 ALE PLE PNLE ASR ALE PLE PNLE ASR ALE PLE PNLE ASR

Fig. 3 The ancestral states of bats versus modern foraging categories. The ancestral states (maximum likelihood estimate of the root node) of six continuous traits considered in this study are shown with 95% confidence intervals. The tree was re-rooted at each internal nodes and contrasts state at the root was computed each time. AS estimate at the root compared to extant foraging categories for: a body mass (N = 183), b eye mass (N = 183), c neocortex mass (N = 149), d superior colliculus mass (N = 84), e inferior colliculus mass (N = 84), and f auditory nucleus mass (N = 84). The ancestral state range of eye mass and non-auditory brain regions (b-d) suggest an increase in pteropodids, while those of the auditory regions (e, f) suggest a basic auditory brain design has been conserved in all bats. We found that the auditory regions (i.e., inferior colliculus, auditory nucleus) were the only brain regions that did not differ between the ancestral bat and today’s species (see also Supplementary Fig. 1), supporting the notion that the ancestral bat had an auditory brain sufficient for echolocation the common ancestor. This trend is largely due to enlargement of investment, and information use48. Strikingly, although we con- – the olfactory bulb, hippocampus, and neocortex in phytophagous firmed that phytophagous species have relatively larger brains40 bats41,43 (Fig. 3; Supplementary Fig. 1). Further, our analyses 42 and non-auditory brain regions than today’s predatory demonstrate the ancestral bat had a relatively larger brain than bats41,43, and than the ancestral bat (Supplementary Table 2; some, but not all, extant predatory bat lineages, perhaps recon- Supplementary Fig. 2), we found that the ancestral bat’s auditory ciling a current point of contention37,46 (Supplementary Fig. 2). brain regions were of the same relative size as in extant predatory Our results also support the hypothesis that this bat used multi- bats and had auditory regions roughly the same absolute size as harmonic (MH) echolocation calls, and thus that constant- those found in today’s LE bats (Fig. 3; Supplementary Table 1; frequency (CF) and dominant-harmonic (DH) call designs are Supplementary Fig. 1). derived states5,12,14,29 (Fig. 2b). This outcome supports our hypothesis that the ancestral bat Based on this hypothetical framework, we now consider three had sufficient auditory powers that could plausibly allow for a hypotheses about the evolution of bat echolocation. Specifically, sonar solution in aid of detecting night flying insects. However, we investigate what potential auditory opportunities and visual this bat would have almost certainly possessed a sonar system less constraints may have characterized this common ancestor, and sophisticated than those of today’s predatory bats. First, because the past and present relationships between these senses in bats. A ~65 million years of evolutionary refinement have since diet of night-flying insects is thought to have constrained the elapsed5,12,19,22. Second, sonar performance should also have ancestral bat (and indeed most predatory bats today) to a small improved because the night-active insects have since become body size47. Based on our results, we contend that this nocturnal better at evading pursuit and thus represent ~65 million years of ancestor had an auditory system sufficient to afford echolocation, selective pressures on most bats’ sonar systems to effectively track but eyes too small in absolute size—due to the constraints of prey15,19. Indeed, while paleontological evidence suggests that the small skull—to allow successful tracking of night-flying prey35. oldest known fossil bat, the insect-eating Onychonycteris finneyi We also provide evidence that species-specific trade-offs between (~52.5 mya), possessed the tympanal bone connection necessary vision and sonar persist to this day. for sonar target ranging11, the cochlea suggests frequency dis- To better understand vertebrate brain evolution, it is now crimination and upper frequency sensitivity inferior to that of established that we should consider not only brain and brain most extant LE bats49. region size in relative terms, but in terms of absolute size. This is Our results suggest to us that pteropodids have apparently because absolute size better reflects processing power, neural maintained auditory brain regions of the same absolute size as the

6 NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x ARTICLE

a b 4 6 3

5 2

1 4 Log eye mass (mg)

0 MH bats Log neocortex mass (mg) CF bats 3 DH bats –1 8 9 10 11 12 8 9 10 11 12 Log body mass (mg) Log body mass (mg)

Fig. 4 Phylogenetically informed linear regressions of eye mass and neocortex on body mass by call type. Phylogenetic generalized least square models showing the regressions of log-transformed a eye mass (N = 162) and b neocortex (N = 162) on body mass while accounting for the phylogenetic non- independence between data points. The calls of laryngeal echolocating bats were categorized as (i) constant frequency (CF), (ii) multi-harmonic calls (MH), (iii) frequency modulated calls dominated by the fundamental harmonic (DH) and are shown separately on the plots

exhibit a rapid increase in cochlea size, similar to laryngeal echolocators and faster than other mammals8. They are also more sensitive to high frequency sounds than are similar-sized terres- 6 trial mammals9,26. Indeed, the echolocation calls of most bats have peak frequencies between 20–60 kHz12,15,50, well within most pteropodids’ auditory limits9,51. Further, genetic vestiges suggest ancient biosonar abilities in the pteropodids10. 4 While LE is unknown in extant pteropodids–as is the case for echolocation of any kind in almost all ~200 pteropodid species–the biosonar-based orientation abilities of the tongue-clicking pter- opodid, Rousettus aegyptiacus, have recently been recognized as being more sophisticated than previously thought25.Furthermore, 2 more rudimentary echo-based orientation has now been experi- Log eye mass (mg) mentally supported in at least two other pteropodid genera, based Pteropodidae on wing clicks potentially used in nature for finding suitable Phyllostomidae roosting places in dark caves52,53. Taken together, all of the above Emballonuridae 0 Rhinolophidae suggests that not only was LE lost, rather than never present, in the Vespertilionidae Pteropodidae, but that the foundations for chiropteran echoloca- tion may not have regressed entirely and instead remain available 8 9 10 11 12 13 44 to be built upon in this lineage. Indeed, this has, perhaps, happened several times already (see Fig. 3 in ref. 53). Log body mass (mg) Our results also demonstrate that echolocation may have ori- Fig. 5 Phylogenetically informed linear regressions of brain and eye mass ginated first in the progenitors of bats, and only rarely in any on body mass by family. Phylogenetic generalized least square models vertebrate group thereafter26,54, not simply because they were showing the regressions of log-transformed eye mass (N = 183) on body pre-adapted for a sonar solution, but also because they were mass accounting for phylogenetic non-independence among data points. constrained by a small body37,47 (Fig. 3), and thus skull and orbit The 5 most speciose families of bats are shown separately on the plots. The size35, from instead realizing a vision-based solution. That is, pteropodids have the largest absolute eye size while the predatory while our AS reconstruction indicates that the ancestral bat had emballonurids have the largest eyes among the laryngeal echolocating bats. relatively and absolutely larger eyes (~3 mm diameter) than most The smallest eyes are found in the more advanced echolocating extant LE bats (Fig. 3; Supplementary Fig. 2; Supplementary rhinolophids and vespertilionids, suggesting that there may be an Data 1), these same results reveal that their eyes were both echolocation-vision trade-off even among predatory species. Repeated relatively and absolutely smaller than those of all extant pter- from last part of our response to AQ3 above:You are correct, thanks for opodid species, including those pteropodid species smaller in spotting it -- there is no (a) and (b) in Figure 5 and we should not have body size than the ancestral bat (i.e., all extant pteropodid species written "... log-transformed (a) brain (N=183) and (b) eye (N=183) on body have eyes >5 mm diameter, while the smallest species weigh ~15 mass" in the first unbolded sentence of the figure heading. This should g; Fig. 3; Supplementary Figs. 1, 2). As we outline below, verte- instead read simply, "log-transformed eye mass (N=183) on body mass". brate eyes of the size estimated for the ancestral bat would be, We had brains in an earlier two-panel version and after cutting the brain/ then and now, too small to allow for the successful aerial pursuit body panel, forgot the cut down the figure heading completely. of even undefended flying insects at night. For two closely related vertebrates of similar size, one noc- common ancestor and extant LE bats (Fig. 3; Supplementary turnal and the other diurnal, relatively larger eyes in the former is Table 1; Supplementary Fig. 1). This lends support to the the norm and indicative of greater investment in vision to be able hypothesis that LE was lost, rather than never present, in this to acquire enough light to see adequately at night55,56. For phytophagous lineage (Fig. 2a), as do several other lines of evi- example, crepuscular aerial insectivorous birds not only have dence. During prenatal cochlear development, pteropodids

NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications 7 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x disproportionately larger eyes but also have relatively larger skulls Methods than otherwise similarly sized diurnal aerial insectivorous Species categorization and brain data. Bat species were classified according to birds55,57, and have average body weights of ~50 g or more47. two systems. First, as (i) a pteropodid species, (ii) a phytophagous laryngeal ’ echolocating (LE) species (roughly two-thirds of extant phyllostomid species), or Thus, we suggest the ancestral bats skull may have been too small (iii) as animal-eating LE bats (all remaining species, representing all families except to afford eyes large enough to allow sufficient sensitivity and the Pteropodidae). Diet and foraging strategies were assigned based on behavioral – resolution to guide and control flight at low light intensities and observations from the literature43,44,64 66. That is, we did not further subdivide successfully track and capture flying insects. Under this scenario, predatory bats into gleaners and trawlers because all predatory bats are apparently able to take prey on the wing15,19,44,67, but those species that have been reported to the reduction in relative eye size in extant LE bats as compared to also glean and trawl prey may reflect observation and reporting biases44. ancestral bat would reflect a greater reliance on more sophisti- We also categorized each bat species to one of the four sensory categories put cated echolocation over evolutionary time and a reduced reliance forth by Griffin5: (i) bats that do not use LE (i.e., pteropodids), (ii) LE bats that only on vision (Fig. 3, Supplementary Fig. 1). The loss of a functional produce multi-harmonic calls (MH bats), (iii) LE bats that can produce steeply downward sweeping calls with most energy in the fundamental harmonic (DH bats), SWS opsin gene in CF bats (Supplementary Table 7; Supple- and (iv) LE bats which produce constant frequency call designs (CF bats)5,6,12,21.We mentary Fig. 3), likely resulting in monochromatic rather than also categorized bats as roosting internally or under exposed conditions. For the list dichromatic vision in these sophisticated echolocators36, supports of species and categories, see Supplementary Data 1. Last, for those bat species in the the plausibility of this viewpoint. phylogeny38 for which reliable genetic visual pigment data exist, we categorized species as having either functional or non-functional short-wavelength sensitive Conversely, the sensory divergence of the pteropodids away – (SWS) opsin genes36,68 71 (see Supplementary Table 7). from early LE and towards a primarily vision-based solution The absolute and relative sizes of the brain and brain regions reflect cognitive reflects a transition from an insect to plant-based diet. This shift and spatial memory performance and reflect the degree to which different sensory to relatively larger, energy-rich, stationary food times would have modalities are relied upon to acquire environmental information. We compared allowed for larger bodies, skulls, and eyes, and selected for larger total brain mass and six brain regions among bats with different echolocating abilities, foraging strategies, and diets: the neocortex, hippocampus, olfactory bulb, brain regions associated with vision, olfaction, and spatial superior colliculus, inferior colliculus, and auditory nucleus. The superior colliculus 45 memory . LE likely regressed due to physiological cost and lack and olfactory bulb are primarily involved in tracking visual and processing odor of stabilizing selection, given the reduced benefit of sonar for stimuli, respectively72. The inferior colliculus and auditory nucleus are primarily 37,72 locating stationary ripe fruit and flowers relative to detecting and devoted to processing auditory information . The hippocampus plays an important role in memory and spatial information processing and the neocortex in tracking small moving insects. Notably, almost no phytophagous higher order cognition and complex stimuli perception72. Mass, brain and brain bat is known to use derived (i.e., CF or DH) echolocation calls. region masses were taken from ref. 72. The pteropodid Rousettus aegyptiacus is a tongue-clicking echo- 58 locator, while all phytophagous phyllostomids, but one , use MH Phylogenetic signal. Closely related species tend to be more similar to one another call designs (Supplementary Data 1). than to those more distantly related, thus species data are not statistically inde- Among predatory bats, all of which laryngeally echolocate, we pendent73. We used phylogenetic comparative methods to control for this non- found that those that produce strictly MH calls had relatively independence. We estimated the degree to which phylogeny predicts the pattern of covariance among species with Pagel’s lambda39 and the Shi and Rabosky38 tree. larger eyes than did DH and CF bats (Fig. 4, Supplementary All subsequent analyses were phylogenetically informed. Table 6). Our results and the conclusions of researchers before us5,12,14,21 indicate that MH calls most closely resemble those of Continuous and categorical AS reconstruction. We used phytools (v. 0.5–38) to the ancestral bat (Fig. 2b) and are closest in structure to those of 74 12,14 reconstruct ASs for all log-transformed continuous variables, which we then anti- non-echolocating terrestrial mammals . Our results therefore logged. The confidence intervals of the ancestral estimates for each variable were suggest that although absolute and relative eye size has decreased then compared to species-level modern categories (Fig. 1). We also used AICc in all extant lineages of predatory bats as compared to the scores to determine the most appropriate model of rate evolution and with phytools – 74 common ancestor (Fig. 3, Supplementary Fig. 1, Supplementary (v. 0.5 38), estimated the scaled likelihoods of each AS at the root node for our three foraging categories, four call type categories, two roost categories and for the Fig. 2), relative eye size has decreased least in MH bats and most functionality of the SWS opsin gene. The probabilities of these ancestral character in DH and CF bats. Our analyses suggest that this difference is estimates have been overlain on the phylogenies in Fig. 2 and Supplementary Fig. 3. not accounted for by roost preference, and suggest that the exclusively predatory emballonurids have eyes at least as large as Eye size estimation. To estimate eye size without sacrificing bats, eyes were those of phyllostomids (Fig. 5). modeled for those species that (i) were found in ref. 72, (ii) occurred in the recent While relatively and absolutely larger eyes in MH bats suggest comprehensive molecular phylogeny of Shi and Rabosky38, and (iii) for which the better night vision, DH and CF call designs are not only derived Royal Ontario Museum (ROM, Toronto, Canada) or the Natural History Museum fl of Denmark (Copenhagen) had at least one intact adult skull. This resulted in but may be superior for detecting and tracking ying prey. DH 183 species (name-matched using the taxonomy and species binomials found in bats (and to a lesser extent, CF bats) can adjust their sonar beam Wilson and Reeder75 representing 18 of 21 chiropteran families). Plasticine balls shape to suit habitat and task (reviewed in ref. 19), while MH bats were made by hand to comfortably fit into the orbit of each skull, near the optic apparently cannot28,30. Additionally, only CF bats, and perhaps nerve foramen (using at least one male and one female whenever possible), as described and validated by Brooke and colleagues55. Ball diameter was measured some DH bats, use acoustic glints resulting from echoes from fi ’ fl using digital calipers and used as a proxy of species-speci c eye diameter to esti- insects apping wing to detect targets in cluttered habitat mate eye mass55. We further confirmed the validity of this non-lethal means of eye 29 (reviewed in ref. ). With respect to production, DH and CF call size estimation using two other methods (see below), and compared all estimates designs require laryngeal specializations for harmonic suppres- with fresh eye diameters from the literature. sion, steep downward frequency sweeps and, in the case of CF First, using the same skulls, we took photographs (using a Nikon D40x digital 59 SLR camera) of their dorsal surface. The maximum zygomatic breadth (ZB) and bats, constant frequency components . Further, the cochlea of the least interorbital breadth (IOD) were measured and the difference between DH and CF bats are more specialized relative to non-echolocating these measures was used as an alternative proxy of eye size. Second, we – mammals than are those of MH bats60 62. Interestingly, the only photographed the eyes of intact alcohol-preserved specimens at the ROM (150 of predatory bat known to “shut off” echolocation while hunting 183 species). Whenever possible, at least one male and one female were used. The 63 horizontal palpebral aperture was used as a proxy for eye length, measured as the under bright moonlight is the MH bat, Macrotus californicus . distance between the medial and lateral canthi. We exported all photos to Image J Thus, a trade-off between echolocation and vision in bats v. 1.49 (National Institutes of Health, USA) and took measurements three times for apparently endures to this day, and is not accounted for only by each specimen to obtain a mean value for each species, from which we estimated diet, but extends to bats that continue to hunt flying insects on diameter. Last, we took the axial lengths of fresh eyes for 33 species from the literature32 and compared to the three potential proxies for eye size (i.e., Plasticine the wing under the cover of night. models, the difference between the ZB and IOD, and eyelid lengths from wet specimens). For eye and skull measurements, specimen numbers, and museum collections, see Supplementary Table 8.

8 NATURE COMMUNICATIONS | (2018) 9:98 | DOI: 10.1038/s41467-017-02532-x | www.nature.com/naturecommunications NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-02532-x ARTICLE

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